The mucus clearance system of the lung represents a key innate immune system that protects the airway surface against constant exposure to inhaled infectious and noxious particles. However, abnormal clearance of mucus by cilia beating and cough represents a significant contributor to the pathogenesis in so-called muco-obstructive lung diseases, including cystic fibrosis, asthma, and chronic bronchitis. Despite the role that reduced clearance of mucus plays in patients with muco-obstructive lung diseases, there are large gaps in our knowledge of how abnormal mucus produces such mucostasis. In order to understand the root causes of the pathology and develop effective therapies to treat such diseases, it is necessary to understand the fundamental mechanisms involved in regulating mucus clearance in health and understand how is it affected by disease. Therefore, the goal of this project is to answer a number of unresolved questions regarding the mucus clearance system. In the first specific aim, we seek to understand what role airway mucins, the large biopolymers which give mucus its gel-like properties, play in facilitating efficient mucus clearance in health. Here, we will use novel in vitro cilia- and cough- mediated clearance assays to test the hypothesis that airway mucins are vital for efficient mucus clearance out of the lung, as a result of interactions between mucins and the airway cell surface. Studies will also identify which mucin domains are involved in this interaction. In the second aim, we will address another outstanding question of whether higher-order multimerization of mucins, to very long polymers, is required for efficient mucus transport. This question has clinical relevance as disulfide bond reducing agents, which ?cleave? long-chain mucins and reduce the elastic properties of mucus, have been proposed as a mucolytic therapy for lung diseases. In this aim, we will test the hypothesis that mucin polymerization is required for efficient cilia- and cough-mediated mucus transport by facilitating the clearance of mucus transport across regions of the airways with poor cilia beat coordination or devoid of ciliated cells, as a result of airway damage associated with gastric aspiration, cigarette smoking, and certain viral infections. In the third aim, we will test the hypothesis that mucin/cell surface interactions are abnormal in diseases associated with hyperproduction of secreted MUC5AC mucin, as a result of Th2 cytokine signaling. Specifically, we will test the hypothesis that chronic IL-13-mediated increases in MUC5AC expression can increase the strength of the cilia-mucus interactions, generating a more adherent, hard to clear, mucus layer. To assess the impact of disease-related increases in MUC5AC expression we will utilize novel mucus adhesion and friction testing devices which quantifies the adherence of mucus to the airway surface and resistance of mucus propulsion. The testing of these hypotheses will be critical to address many gaps in our knowledge about mucus clearance system functions in health, why it is dysfunctional in disease, and to identify targets for approaches to restore/accelerate mucus clearance in persons with muco-obstructive lung diseases.